Variation in the Exon 3–4 Region of Ovine KRT85 and Its Effect on Wool Traits

Simple Summary α-keratins are the main structural proteins in the cortex of wool fibres. Variation in these proteins affects the structure and fibre characteristics of wool, making keratin genes good candidates for the development of gene markers to selectively breed for better wool. Variation was found in the keratin gene KRT85, with the presence of the A variant being associated with an increase in greasy fleece weight and clean fleece weight, and the presence of B associated with an increased wool prickle factor. This may assist in the development of gene markers to assist in sheep breeding. Abstract α-keratins are structural proteins in the cortex of wool fibres and assemble in an organized fashion into keratin intermediate filaments. Variation in these keratin proteins affects the structure and characteristics of wool fibre, making keratin genes ideal candidates for the development of gene markers that describe variations in wool traits. A region of KRT85 spanning exon 3–4 (including the entire exon 3, intron 3, exon 4 and part of intron 4) was investigated. Two banding patterns defining two variant sequences (A and B) were observed in this region, and these were characterised by the presence of two single nucleotide polymorphisms. The effect of this variation in the exon 3–4 region of KRT85 on wool traits was investigated in 463 Merino × Southdown-cross lambs. The frequencies of these two variants in these sheep were 55.6% and 44.4%, respectively. Three different genotypes were observed with frequencies of 32.6%, 46.1% and 21.3% for AA, AB and BB, respectively. The presence of A was associated with an increase in greasy fleece weight and clean fleece weight, while the presence of B was associated with an increased wool prickle factor. These findings should be replicated in a broader range of sheep breeds to determine whether the associations are robust and to clarify whether the observed effects are attributable to breed differences or to gene effects themselves.


Introduction
The "hard" α-keratins contribute to the formation of keratinized structures in mammalian nails, hair and claws [1], and they are the major proteins in wool fibres.These keratins have been classified into two families, type I and type II keratins, with the type I keratins being acidic proteins and the type II keratins being basic-to-neutral proteins.To date, eleven functional type I and six type II keratin genes (KRTs) have been identified in human hair [1,2].In sheep, 17 keratin genes have been identified, but their composition differs based on the absence of KRT37 in the type I family and the presence of KRT87 in the type II family [3].
Previous studies have suggested that the QTLs affecting wool traits are located on chromosome 3, with markers observed for greasy fleece weight (GFW) and the coefficient of the variation of fibre curvature (CVFD) [8][9][10].However, only a small number of studies have described genetic variation in the chromosome 3 keratin genes.McLaren et al. (1997) reported two variants of KRT83 using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) typing method [11], and nine variants of KRT87 (formerly KRT2.13) were reported by McKenzie et al. [12].One, two and five variants have been described in the KRT83 promoter, exon 2 and exon 3-4 regions using a PCR single-stranded conformational polymorphism (PCR-SSCP) approach, and the variation in the KRT83 exon 3-4 region was reported to be associated with the mean fibre diameter (MFD) [13].
In this study, PCR-SSCP was used to investigate sequence variation in the exon 3-4 region of KRT85, and the associations, if any, that may occur between KRT85 sequence variations and various commercially important wool traits.

Sheep Blood and Wool Samples
Approval for the present study was obtained from the Lincoln University Animal Ethics Committee under the provisions of the Animal Welfare Act 1999 (New Zealand Government).Initially, 300 test panel sheep from 26 farms, including Merino sheep (n = 100), New Zealand (NZ) Romney sheep (n = 100) and white Dorper sheep (n = 100), were screened to search for variation in the exon 3-4 region of KRT85.Subsequently, 463 Southdown × Merino-cross lambs from seven sire lines were used for the association studies.Blood samples for all the sheep were collected onto FTA cards (Whatman BioScience, Middlesex, UK), and then genomic DNA was extracted from the dried blood spots using a two-step procedure [14].
Wool samples were taken at 12 months of age from the mid-side of the sheep at shearing, and the entire fleece was weighed to give the GFW.Key commercial wool traits were then measured from the samples by the New Zealand Wool Testing Authority Ltd. (NZWTA, Napier, New Zealand) using the International Wool Testing Organization (IWTO)'s standardized methods (https://iwto.org/wool-supply-chain/wool-testing/; accessed 2 July 2024).This included the measurement of wool yield (Yield), MFD, fibre diameter standard deviation (FDSD), CVFD, mean fibre curvature (MFC), mean staple length (MSL), mean staple strength (MSS) and prickle factor (PF; the percentage of fibres of a diameter greater than 30 microns).The clean fleece weight (CFW) was calculated from the GFW and yield (CFW (kg) = GFW (kg) × Yield (%)).

PCR Amplification and Genotyping of the KRT85 Exon 3-4 Region
Based on the ovine KRT85 sequence in the Texel sheep Ovine Genome Assembly v3 (GCF_000298735.1,GenBank), two PCR primers (5 ′ -CTGACTCCTCAGCTCCAGC-3 ′ and 5 ′ -CCCTCACCTCATCATAGAG-3 ′ ) were designed to amplify a 347 bp fragment spanning part of intron 2, exon 3, intron 3 and exon 4 of the gene.Amplifications were undertaken in 15 uL reactions that included the genomic DNA in a 1.2 mm spot of blood on the FTA cards, 10× reaction buffer, 0.5 U of Taq DNA polymerase (Qiagen, Hilden, Germany), 250 nM of each primer, 150 µM of each dNTP (Eppendorf, Hamburg, Germany) and a final Mg 2+ concentration of 2.5 mM.The cycling parameters for PCR amplification consisted of an initial denaturation step at 94 • C for 2 min; followed by 35 cycles of 30 s at 95 • C, 30 s at 58 • C and 30 s at 72 • C; and a final extension at 72 • C for 5 min.Amplification was undertaken in S1000 thermal cyclers (Bio-Rad, Hercules, CA, USA).
To genotype the amplicons produced by the PCR amplifications, a PCR-SSCP technique was used.In these analyses, a 0.7 µL aliquot of PCR product was mixed with 7 µL of loading dye (98% formamide, 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene-cyanol), denatured at 95 • C for 5 min, and then cooled rapidly on wet ice.Electrophoreses were carried out in 0.5× TBE buffer in 14% polyacrylamide gels (37.5:1;Bio-Rad) for 18 h at 29 • C and 200 volts.Once reference samples were determined for each variant, they were included on all subsequent gels to facilitate the precise genotyping of all the sheep.All gels were silver-stained according to the method of Byun et al. [15].Amplicons of the exon 3-4 variants were subsequently sequenced using the rapid approach described by Gong et al. [16].

Statistical Analyses
Statistical analyses were carried out and Minitab version 16.General Linear Mixedeffects Models (GLMMs) were used to assess the effect of the presence/absence of KRT85 exon 3-4 variants on wool traits.In all the models, variant presence/absence, gender and birth rank were fitted as fixed factors, and sire line was fitted as a random factor.
Initially, 'single-variant' presence/absence models were created.Subsequently, any variant-wool trait association in the single-variant models with a p-value of <0.200 (and which could therefore potentially impact the wool trait being tested) were factored into 'multi-variant models', an approach that was used by Forrest et al. [17].
The model used was Y jklm = µ + V1 j + V2 k + G l + S m + e jklm , where Y jklm is the phenotypic value for the jklm th sheep, µ is the group raw mean for the trait, V j is the effect of the j th variant (presence and absence), V2 k is effect of the second variant's presence/absence (if factored in), G l is the effect of gender, S m is the effect of the m th sire and e jklm is the random residual effect.Significance was accepted at p < 0.05.

Detection of Variation in Ovine KRT85 in the Exon 3-4 Region
Two PCR-SSCP gel banding patterns were observed with the conditions that were developed for the analysis of the KRT85 exon 3-4 region, and the genotypes of the 300 test panel sheep and 463 Merino × Southdown-cross lambs could be determined.Upon sequencing, two variant sequences of this region of the gene were confirmed in the amplified region.These sequences were named variants A and B, and the sequences of the two variants have been deposited in GenBank and assigned the accession numbers MH910660 and MH910661.
The variant frequencies were 55.6% and 44.4% for A and B, respectively, in the Merino × Southdown-cross lambs.Despite the breed differences in the 300 test panel sheep, only these two variants were detected in that population too.
In the Merino × Southdown-cross lambs, three different genotypes were observed: AA, AB and BB, with frequencies of 32.6%, 46.1% and 21.3%, respectively.However, frequencies are not reported for the test panel sheep as they were selected to ascertain how much genetic variation may exist in this gene region, and not because they were representative of the different breeds.
The sequence difference between A and B was because of the presence of two single nucleotide polymorphisms (SNPs; c.600G/A and c.652+93C/A) in the amplified region of KRT85 (Figure 1), with one SNP located in exon 3 and the other in intron 3. The SNP located in exon 3 was synonymous and would not result in an amino acid change.

Associations between KRT85 Exon 3-4 Variants and Variation in Selected Wool Traits
The presence of A tended to be associated with increased GFW and CFW in the single-variant GLMMs, an association that became significant in the multi-variant GLMM (Table 1).The presence of B was associated with an increased PF (Table 1).

Discussion
Here, we report the identification of variation in the ovine KRT85 exon 3-4 region.Of the two SNPs detected, one occurred in exon 3 and was synonymous, and the other was in a non-coding intron 3 region.Sulayman et al. [18] reported three nucleotide substitutions in a KRT85 exon 3 fragment of Chinese Merino (Xinjiang type) sheep, with one being silent, one creating a nonsense mutation and one creating a synonymous mutation in the

Associations between KRT85 Exon 3-4 Variants and Variation in Selected Wool Traits
The presence of A tended to be associated with increased GFW and CFW in the single-variant GLMMs, an association that became significant in the multi-variant GLMM (Table 1).The presence of B was associated with an increased PF (Table 1).

Discussion
Here, we report the identification of variation in the ovine KRT85 exon 3-4 region.Of the two SNPs detected, one occurred in exon 3 and was synonymous, and the other was in a non-coding intron 3 region.Sulayman et al. [18] reported three nucleotide substitutions in a KRT85 exon 3 fragment of Chinese Merino (Xinjiang type) sheep, with one being silent, one Animals 2024, 14, 2272 5 of 7 creating a nonsense mutation and one creating a synonymous mutation in the protein [18].The variation described in the Chinese Merino seems to be greater than in all the New Zealand sheep tested, despite the diverse origins of these breeds, with the New Zealand Romney having its origin in British Romney Marsh sheep, the Merino coming from Spain, the Southdown being British and the Dorper having South African origins.
While none of the SNPs described here would appear to have major effects on the K85 protein, synonymous nucleotide variations are increasingly being recognized as a factor that can affect protein expression, such as by altering transcription, mRNA half-life, translation, protein stability and protein function.To illustrate this point, a synonymous variant of the human corneodesmin gene (CSDN) caused an increase in mRNA half-life and was associated with a psoriasis phenotype [19], but its mechanism of action could not be ascertained.In both that study and herein, the silent SNPs may be linked to a nucleotide variation upstream or downstream in an extended haplotype that affects the regulation of gene expression.Equally, while the intron variation might be benign, it is now widely understood that introns impact gene expression, mRNA splicing, mRNA stability and can optimise transcriptional efficiency in their host gene.That is, the presence of intronic mutations can affect regulatory expression elements from transcription to translation [20].
The genetic structure of complex traits, especially the number of loci affecting the trait and the distribution of their effects, is still largely unresolved.Accordingly, further studies are required to assess the effect of polymorphisms in both coding and non-coding regions of candidate genes and their association with mRNA levels, translation, protein processing, the activity of biochemical pathways and the tissue(s) in which the gene is functional.Hence, in this study, it might be more prudent to conclude that KRT85 variation would have a functional impact, until proven otherwise.
Sulayman et al. reported an association between a KRT85 exon 3 fragment and wool traits in Chinese Merino sheep, with the JJ genotype sheep having a significantly greater wool crimp score, body size and fibre diameter than the other genotypes [18].The associations detected in the present study are hard to compare with these findings, with variant B of the KRT85 exon 3-4 region only being associated with PF in the single-variant models, and with the two fleece weight traits CFW and GFW in the multi-variant models.Prickle factor is related to MFD (higher-diameter fibres impart more prickle), but no effect was observed with KRT85 variation and MFC, despite crimp and MFC being phenotypically correlated [21].
The observation that both CFW and GFW were associated with variations in KRT85 is consistent with the strong positive correlation reported between these traits [22].It is also notable that variant A, which is associated with CFW and GFW, was also trending towards an association with MSL.Intuitively, one might expect longer fibres to suggest greater wool growth and hence increased CFW and GFW, and, in that respect, Gong et al. have reported that GFW and CFW are both moderately positively correlated with an increased MSL [22].
KRT85 is located on the same chromosome as KRT83, but variation in the exon 3-4 fragment of KRT83 was associated with MFD, whereas variation in the exon 3-4 fragment of KRT85 was found to be associated with increased GFW and CFW.This suggests that the two genes have independent effects.Gong et al. [22] reported no significant correlation between GFW and CFW and MFD, with this supporting the argument that KRT83 and KRT85 function independently.
The associations detected in this study are consistent with the findings that revealed a QTL for GFW on chromosome 3 [23].The effect of variation in the KRT85 exon 3-4 fragment on wool traits is also similar to that of KRTAP1-2 [22], KRTAP6-1 [24] and KRTAP22-1 [25], despite these KRTAPs being located on different chromosomes.Once again, this suggests that wool traits are not the consequence of the action of individual KRTs or KRTAPs [26], but instead reflect the coordinated expression of many genes.